Abstract Infections from opportunistic bacterial pathogens such as Staphylococcus aureus place a tremendous burden on our healthcare system. The human skin and mucosal surfaces are coated with commensal bacteria that make up the healthy microbiome, and this natural protective layer is important for preventing infections. There is growing evidence that extensive interactions between commensal bacteria occur as they colonize a specific niche and compete for resources. One strategy the bacteria can employ to gain an advantage is to release molecules that negatively impact neighbors. There are growing reports that targeting quorum-sensing could be an effective strategy to reduce fitness and in turn slow growth of a competitor. S. aureus persistently colonizes 20% of the healthy adult population, and this opportunistic pathogen uses quorum-sensing system to control the expression of enzymes, toxins, and immunomodulatory proteins that are essential to spread through tissues and cause disease. This regulatory system is controlled by the production and sensing of a secreted cyclic peptide signal, also called an autoinducing peptide or AIP. In our preliminary studies, we discovered that a commensal staphylococcal strain, S. caprae, releases an AIP signal that competes with all S. aureus strains, including clinical methicillin-resistant S. aureus (MRSA) isolates. Using mass spectrometry, we identified the AIP structure, and this AIP prevents MRSA quorum-sensing activation and skin infection progression in a mouse model. We hypothesize that commensal Staphylococci release AIP signals to compete with S. aureus to gain a competitive colonization advantage. This hypothesis is in part based on our previous findings that a functional quorum-sensing system is necessary to colonize the host. Therefore, we propose that the purpose of the AIP cross-talk could be to gain a fitness advantage in the colonization environment. In Aim 1, we will investigate the mechanism of quorum-sensing inhibition by commensal Staphylococci. Besides S. caprae, we have identified five additional staphylococcal strains that inhibit MRSA quorum-sensing, and we will employ our mass spectrometric strategy to identify the corresponding AIP structures. We will confirm the AIP structural assignment and test each of them in MRSA quorum-sensing reporter assays and toxin production tests. In Aim 2, we will determine the ability of commensal AIPs to prevent MRSA infection using a mouse model. We will monitor MRSA agr activation using bioluminescence with real time mouse imaging in the presence of inhibitory AIPs, and evaluate the ability of these AIPs to prevent development of a MRSA skin infection. Finally, in Aim 3, we will assess the impact of commensal Staphylococci on MRSA colonization. Using a developed mouse skin colonization model, we will assess MRSA colonization of living skin in the presence of varying levels of inhibitory AIPs, and perform competition assays between commensal Staphylococci and MRSA. Understanding how commensal bacteria can reduce the colonization and infection potential of problematic pathogens will improve our knowledge of the microbiome's contribution to human health.